JP4718005B2 - Thermostable glucoamylase - Google Patents

Description

【００７４】
実施例３．ティー・エメルソニイのグルコアミラーゼのＮ末端のアミノ酸配列の
決定
ティー・エメルソニイのグルコアミラーゼのＮ末端のアミノ酸配列を、ＳＤＳ−ＰＡＧＥと、ＰＶＤＦ膜への電気泳動的転写によって決定した。ペプチドは、リシル特異的プロテアーゼによって開裂させることによって、還元しかつＳ−カルボキシメチル化させたグルコアミラーゼから誘導したペプチドであった。得られたペプチドを分画し次にＲＰ−ＨＰＬＣを使って再び精製してから、Ｎ−末端の配列を決定した。
【００７５】
Ｎ−末端の配列（配列番号：１）：
Ala Asn Gly Ser Leu Asp Ser Phe Leu Ala Thr Glu Xaa Pro Ile Ala
Leu Gln Gly Val Leu Asn Asn Ile Gly
ペプチド１（配列番号：２）：
Val Gln Thr Ile Ser Asn Pro Ser Gly Asp Leu Ser Thr Gly Gly Leu
Gly Glu Pro Lys
【００７６】
ペプチド２（配列番号：３）：
Xaa Asn Val Asn Glu Thr Ala Phe Thr Gly Pro Xaa Gly Arg Pro Gln
Arg Asp Gly Pro Ala Leu
ペプチド３（配列番号：４）：
Asp Val Asn Ser Ile Leu Gly Ser Ile His Thr Phe Asp Pro Ala Gly
Gly Cys Asp Asp Ser Thr Phe Gln Pro Cys Ser Ala Arg Ala Leu Ala
Asn His Lys
【００７７】
ペプチド４（配列番号：５）：
Thr Xaa Ala Ala Ala Glu Gln Leu Tyr Asp Ala Ile Tyr Gln Trp Lys
ペプチド５（配列番号：６）：
Ala Gln Thr Asp Gly Thr Ile Val Trp Glu Asp Asp Pro Asn Arg Ser
Tyr Thr Val Pro Ala Tyr Cys Gly Gln Thr Thr Ala Ile Leu Asp Asp
Ser Trp Gln
Ｘａａは指定することができなかった残基を意味する。
【００７８】
実施例４．全長のティ・エメルソニイのグルコアミラーゼ
配列番号：７に示す全長のティ・エメルソニイのグルコアミラーゼのアミノ酸配列を標準の方法を使って同定した。
【００７９】
実施例５．タラロミセス・エメルソニイのグルコアミラーゼの遺伝子のクローン
化と配列決定
タラロミセス・エメルソニイＡＭＧの遺伝子の、ＰＣＲでクローン化する部分。
タラロミセス・エメルソニイＡＭＧをクローン化するために、下記表２に示す縮重されたプライマーを、ＡＭＧ遺伝子の部分をＰＣＲで増幅するために設計した。
【００８０】
【表２】

【００８１】
タラロミセス・エメルソニイ由来のゲノムＤＮＡを、標準の方法でつくったプロトプラストから調整し（例えば、Christensen ら、Biotechnology 、６巻1419〜1422頁1989年参照）し、ＰＣＲ反応の鋳型として使用した。Ｔａｑ−ポリメラーゼ２．５単位、エイ・オリゼのゲノムＤＮＡ 100ng 、50mM KCl、10mMトリス−HCl(pH8.0)、1.5mM MgCl₂ 、250nM の各dNTP、ならびに100pM の下記各プライマーのセット：102434／117360，102434／117361，102435／117360，102434／117361，102434／127420および102434／127420を含有する 100μｌ容積で、増幅反応を行った。
【００８２】
増幅は、Perkin-Elmer Cetus DNA Termal 480 で実施し、９４℃で３分間の１サイクル、これに続く、９４℃で１分、４０℃で３０秒および７２℃で１分の３０サイクルで構成された増幅であった。ＰＣＲ反応102434／117360だけが産物を提供した。1400, 800, 650および５２５bpのサイズの四つのバンドが検出された。これら四つのバンドをすべて精製し、ベクターｐＣＲ（登録商標）２．１〔Invitrogen（登録商標）〕中にクローン化した。各バンド由来のいくつかのクローンの配列を決定し、エイ・ニガーＡＭＧと配列を比較した結果、６５０bpのバンド由来のクローンがタラロミセス・エメルソニイＡＭＧのＮ末端部分をコードすることが明らかになった。このクローンをpJaL497 と命名した。
【００８３】
前記遺伝子をより多く得るため、特異的プライマー（123036：5'-GTGAGCCCAAGTTCAATGTG-3'( 配列番号：１５）を、クローンpJaL497 の配列で製造した。プライマーのセット123036／127420をタラロミセスのゲノムＤＮＡにＰＣＲを用いて、1500bpの単フラグメントを得た。そのＰＣＲフラグメントをベクターpCR2.1中にクローン化して配列を決定した。配列を決定することによって、そのクローンは、タラロミセス・エメルソニイＡＭＧのＣ末端部分をコードすることが確認された。そのクローンをpJaL507 と命名した。
【００８４】
ゲノムの制限地図の作成およびゲノムクローンのクローン化
上記二つのクローンpJaL497 とpJaL507 とで、前記ＡＭＧ遺伝子の約９５％を占めていた。前記ＡＭＧ遺伝子の欠けている部分（missing part）をクローン化するため、これら二つのＰＣＲフラグメントを、プローブとして、単一の制限酵素またはいくつもの制限酵素を組み合わせたもので消化されたタラロミセス・エメルソニイのゲノムＤＮＡのサザンブロットに使用することによって、ゲノム制限地図を作成した。この地図は、タラロミセス・エメルソニイＡＭＧの遺伝子が、二つのEcoRI フラグメントに位置し、それぞれ約５．６kbと約６．３kbであることを示している。
【００８５】
タラロミセス・エメルソニイのゲノムＤＮＡをEcoRI で消化し、大きさが４〜７kbのフラグメントを精製し、これを、製造業者の使用説明書（Stratagene）に記載されているように使用して、λ2APII 中に部分ゲノムライブラリーを構築した。そのライブラリーを、第一に、pJaL497 由来の０．７kbのEcoRI フラグメント（ＡＭＧ遺伝子のＮ末端の¹/₂ をコードする）をプローブとして用いて選別して、ＡＭＧ遺伝子の出発物(start）を得た。クローン１種を得て、pJaL511 と命名した。pJaL507 由来の０．７５kbのEcoRV フラグメント（ＡＭＧ遺伝子のＣ末端の¹/₂ をコードする）をプローブとして使用して、前記ライブラリーの第二の選別を行って、ＡＭＧ遺伝子のＣ末端を得た。１種のクローンが得られ、pJaL510 と命名した。
【００８６】
タラロミセス・エメルソニイＡＭＧの遺伝子の配列分析
プラスミド類：pJaL497, pJaL507, pJaL510 およびpJaL511 ならびにそれらのサブクローンの配列を、ｐＵＣに対する標準の復帰(reverse）プライマーと前進(forward）プライマーを用いて決定することによって、ＡＭＧ遺伝子の配列を得た。残りのｇａｂは、特異的なオリゴヌクレオチドをプライマーとして使用して閉じた。
【００８７】
上記配列を、アスペルギルス属の真菌中のイントロンのコンセンサス配列およびエイ・ニガーのＡＭＧの配列と比較することによって、潜在イントロン（potential intron）が見出された。タラロミセス・エメルソニイＡＭＧのヌクレオチド配列は、６１８のアミノ酸のタンパク質をコードし、それぞれ、５７bp、５５bp、４８bpおよび５９bpの四つのイントロンで遮断された(interrupt）オープンリーディングフレームを有している。そのヌクレオチド配列（イントロン含有）と推定アミノ酸配列を図５に示す。
【００８８】
また、そのＤＮＡ配列（イントロン含有）を配列番号：３３に示し、そしてタラロミセス・エメルソニイＡＭＧの配列（１〜２７のシグナル配列含有）を配列番号：３４に示す。前記推定アミノ酸配列を、エイ・オリゼＡＭＧとエイ・ニガーＡＭＧと比較したところ、同一度（identity）がそれぞれ６０．１％と６０．５％であった。図６に示すアミノ酸配列の配列状態は、タラロミセスＡＭＧが、触媒ドメインと澱粉結合ドメインの間に非常に短いヒンジ部を有していることを示している。このことは、エイ・オリゼＡＭＧにも見られる。
【００８９】
実施例６．アスペルギルスのベクターｐＣａＨｊ４８３の構築
pCaHj483の構築を図７に示す。このプラスミドは下記のフラグメントから構築する。
ａ）EcoRI とXbaIで切断されたベクターpToC65（国際特許願公開第WO91／17243 号）。
【００９０】
ｂ）ａｍｄｓ遺伝子(C. M. Corrickら、Gene, 53巻63〜71頁1987年）を保持するエイ・ニデュランス由来の2.7kb のXbaIフラグメント。このａｍｄｓ遺伝子は、真菌を形質転換する際の選択マーカーとして使用される。このamds遺伝子は変形されて、この遺伝子中に通常、存在しているBamHI 部位が破壊されている。この破壊は、プライマー：5'-AGAAATCGGGTATCCTTTCAG-3'(配列番号：１６）を使って、サイレントポイント突然変異を導入することによって行った。
【００９１】
ｃ）エイ・ニデュランスのｔｐｉ遺伝子のｍＲＮＡの５’−非翻訳末端をコードする配列の60bp DNAフラグメントに融合されたエイ・ニガーのＮＡ２プロモーターを保持する０．６kbの EcoRI／BamHI フラグメント。上記ＮＡ２プロモーターは、プラスミドｐＮＡ２（国際特許願公開第WO89／01969 号に記載されている）から単離し、次いで６０bpのｔｐｉ配列に、ＰＣＲによって融合させる。６０bpのｔｐｉ配列をコードするプライマーは下記の配列であった。
【００９２】
5'-GCTCCTCATGGTGGATCCCCAGTTGTGTATATAGAGGATTGAGGAAGGAAGAGAAGTGTGGATAGAGGTAAATTGAGTTGGAAACTCCAAGCATGGCATCCTTGC-3'(配列番号：１７）
ｄ）エイ・ニガーのグルコアミラーゼの転写ターミネーターを保持する６７５bpのＸｂａＩフラグメント。このフラグメントは、プラスミドpICAMG／Term（欧州特許第 0238023号に記載されている）から単離した。
フラグメントｃのBamHI 部位を、フラグメントｄの転写ターミネーターの先頭のXbaI部位に、pIC19Rリンカーを介して接続した（BamHI からXbaI）。
【００９３】
ＡＭＧ発現プラスミドｐＪａＬ５１８の構築
タラロミセス・エメルソニイＡＭＧ遺伝子のコード領域を、下記の二つのオリゴヌクレオチドプライマー、すなわち
プライマー139746：5'-GACAGATCTCCACCATGGCGTCCCTCGTTG-3'（配列番号：１８）、および
プライマー139747：5'-GACCTCGAGTCACTGCCAACTATCGTC-3'(配列番号：１９）
を使用して、ＰＣＲによって増幅した。下線をつけた領域は、タラロミセス・エメルソニイＡＭＧ遺伝子内に存在する配列を示す。クローン化を容易にするため、各プライマーの５’末端に、制限酵素部位を挿入した。プライマー139746はBglIII部位を含有し、そしてプライマー139747はXhoI部位を含有している。
【００９４】
タラロミセス・エメルソニイのゲノムＤＮＡを、ＰＣＲ反応の鋳型として使用した。ＰＣＲ反応は、２．５単位のＴａｑポリメラーゼ、１００ngのｐＳ０２，２５nMの各ｄＮＴＰ、および上記２種のプライマー各々の１０pmolを含有する反応緩衝液（50mM KCl、10mMトリス−HCl(pH8.0)、1.5mM MgCl₂ ） 100μｌの容積内で実施した。
【００９５】
増幅はPerkin-Elmer Cetus DNA Termal 480 で実施したが、９４℃で３分間の１サイクルと、これに続く９４℃で１分、５５℃で３０秒および７２℃で１分の２５サイクルで構成されている。このＰＣＲ反応によって、長さが2099bpの単一のＤＮＡフラグメントが産生された。このフラグメントをBglII とXhoIで消化し、次いでゲル電気泳動法で単離し、精製し、次にBamHI とXhoIで消化したpCaHj483中にクローン化してプラスミドを得、これをpJaL518 と命名した。このように、プラスミドpJaL518 が構築されて、タラロミセス・エメルソニイＡＭＧ遺伝子用の真菌発現プラスミド（図８）が得られた。
【００９６】
アスペルギルス・ニガー菌株、ＳＭ０１１０の構築
１．エイ・ニガーのpyrG遺伝子のクローン化
製造業者の説明書に記載されているようにして、エイ・ニガーBO-1をEMBL4 中に作製した。公開されたアスペルギルス・ニガーの配列（Wilsonら、Nucleic Acids Res., 16巻2339〜2339頁1988年）から設計した、ＤＩＧで標識したオリゴヌクレオチド〔PyrG：5'-CCCTCACCAGGGGAATGCTGCAGTTGATG-3'(配列番号：２０）〕によって、前記ライブラリーを選別した。上記ＤＩＧプローブとハイブリッドを形成した陽性のEMBL4 クローンをBO-1ライブラリーから単離し、次に、pyrG遺伝子を含有する3.9kb のXbalフラグメントをEMBL4 からサブクローン化し次いでpUC118中にクローン化してpJRoy10 を作製した。
【００９７】
２．エイ・ニガーのグルコアミラーゼ（ＡＭＧ）の遺伝子のクローン化
下記のオリゴヌクレオチド950847：5'-CGCCATTCTCGGCGACTT-3'（配列番号：２１）とオリゴヌクレオチド951216：5'-CGCCGCGGTATTCTGCAG-3'（配列番号：２２）〔公開されたアスペルギルス・ニガーの配列（Boelら、EMBO J., 3巻1581〜1585頁1984年）から設計された〕を用いて、エイ・ニガーのゲノムＤＮＡ上で増幅することによって生成させたＤＩＧ標識化ＰＣＲフラグメントで、上記エイ・ニガーのＢＯ−１ライブラリーを選別した。上記ＤＩＧプローブとハイブリッドを形成した陽性のEMBL4 クローンをＢＯ−１ライブラリーから単離し、次にそのＥＭＢＬ４クローンから、ＡＭＧ遺伝子を含有する4.0kb のSpeIフラグメントをサブクローン化し次いでプラスミドpJRoy17aを生成するpBluescriptSkt中にクローン化した。
【００９８】
３．エイ・ニガーＡＭＧの分断カセット（disruption cassette)の構築
pyrGを含有する２．３kbのSpeI−XhoIフラグメントを、pJRoy10 からゲルで単離し、その制限末端にクレノウポリメラーゼを充填した。そのフラグメントを、ＡＭＧ遺伝子内で切断してプラスミドpSM0127 を生成するpJRoy17 のBglIII部位中に挿入した（図９）。pSM0127aの二つのSpeI部位の間に、２．２kbの５’ＡＭＧと２．３kbの３’ＡＭＧが隣接している２．３kbのpyrG遺伝子が入っている。
【００９９】
４．ＡＭＧを得るため分断されたエイ・ニガーの株：SM0110の構築
エイ・ニガーのJRoyP3は、エイ・ニガーBO-1のpyrGの自然突然変異体であり、これを選別して５’−フルオロ−オロト酸（5'-FOA）が入っているプレート上で増殖させた。pyrG遺伝子は、オロチジン５’−ホスフェートカルボキシラーゼをコードするので、その欠失変異体はウリジン要求株として特徴づけることができる。pyrG変異体の同一性は、最少培地での増殖が、エイ・ニデュランスのpyrG遺伝子と相補的であることによって確認された。
【０１００】
２０μｇのプラスミドpSM0127 をSpeIで消化した。そのＤＮＡを０．８％のアガロースゲル上で分割し(resolve）、線状分断カセットからなる６kbをゲルで単離した。その線状ＤＮＡで菌株ＪＲｏｙＰ３を形質転換した。次にＳｐｅＩで消化した２００の形質転換体からゲノムＤＮＡを調製した。前記ゲルで分割したＤＮＡをハイボンドナイロン（hybond nylon）フィルターに移し、ＡＭＧのオープンリーディングフレームからなる非放射性ＤＩＧプローブとハイブリッドを形成させた。分断カセットをＡＭＧ遺伝子座へ入れることによって、野生型の４kb ＡＭＧのバンドが６．３kbまで増大した。この増大は２．３kbのｐｙｒＧ遺伝子によるものである。上記特性を有する一つの形質転換体＃１１０を選別してさらに分析した。
【０１０１】
形質転換体＃１１０を、ＹＰＭ培地が入っている２５mlの振盪フラスコ中で増殖させた。菌株ＢＯ−１と親菌株JRoyP3を、ＡＭＧ産生の対照物として増殖させた。３日後、３０μｌの透明上澄み液を、８〜１６％のSDS PAGE Novexゲル上で泳動させた。ＡＭＧのバンドは形質転換体＃１１０中に全く見られなかったが、ＡＭＧの多数のバンドが、正の対照の菌株BO-1と親菌株JRoyP3に産生した。形質転換体#110をSM0110と命名した。
【０１０２】
アスペルギルス・オリゼおよびアスペルギルス・ニガーにおけるタラロミセス・エメルソニイＡＭＧの発現
JaL228とSM0110の菌株を、Christensen ら、Biotechnology, 6巻1419〜1422頁1988年に記載されているようにしてpJaL518 で形質転換した。一般に、エイ・オリゼの菌糸体を富栄養ブロス中で増殖させた。その菌糸体を、前記ブロスから濾過によって分離した。酵素製剤Novozyme（登録商標）（Novo Nordisk）を、リン酸ナトリウムによってpH５．０に緩衝された１．２Ｍ ＭｇＳＯ₄ などのような浸透によって安定化する緩衝液中の前記菌糸体に添加した。
【０１０３】
得られた懸濁液を、撹拌しながら、３７℃で６０分間インキュベートした。プロトプラストをミラクロスで濾過して菌糸体の破片を除いた。そのプロトプラストを収穫して、STC(1.2Mソルビトール、10mM CaCl₂、10mMトリス−HCl pH7.5)で２回洗浄した。最後に、プロトプラストを、 200〜1000μｌのＳＴＣ中に再懸濁させた。
【０１０４】
形質転換を行うため、５μｇのＤＮＡを 100μｌのプロトプラスト懸濁液に添加し、次に 200μｌの PEG溶液（60％PEG4000 、10mM CaCl₂、10mMトリス−HCl 、pH7.5)を添加して、その混合物を室温で２０分間インキュベートした。プロトプラストを収穫して、１．２Ｍソルビトールで２回洗浄した。最後に、そのプロトプラストを、２０μｌの１．２Ｍソルビトール中に再び懸濁させ、選択プレート上にプレートし〔最少培地＋10ｇ／ｌのBacto-Agar（Difco)〕、次いで３７℃でインキュベートした。３７℃で３〜４日間、増殖させると、安定な形質転換体が、激しく増殖して胞子を形成するコロニーとして出現する。形質転換体は胞子で２回単離された。
【０１０５】
振盪フラスコ内の100ml のＹＰＭ培地（２ｇ／ｌの酵母エキス、２ｇ／ｌのペプトンおよび２％のマルトース）中で、形質転換体を、３０℃で４日間増殖させた。上澄み液を、先に述べたようにしてＡＭＧ活性について試験し、次いでSDS PAGEゲルで分析した（図１０）。
【０１０６】
実施例７．酵母内で発現させるために行う、タラロミセス・エメルソニイＡＭＧ
のＤＮＡ配列からの四つのイントロンの除去
各エキソンについて、隣りのエキソンに対しオーバーラップ部分を含有するプライマーを用いて、ＰＣＲ反応を行った。Tal1とTal4は、酵母ベクターpJSO026 とのオーバーラップ部分を含有している。エキソン１：Ｔａｌ１は５’プライマーとして使用し、そしてTal5は３’プライマーとして用い、そしてＡＭＧをコードするゲノム配列を鋳型として使った。エキソン２：Ｔａｌ６を５’プライマーとして使い、そしてTal7を３’プライマーとして使用し、そしてＡＭＧをコードするゲノム配列を鋳型として使用した。
【０１０７】
エキソン３：Ｔａｌ８を５’プライマーとして使用し、そしてTal9を３’プライマーとして使用し、そしてＡＭＧをコードするゲノム配列を鋳型として使用した。エキソン４：Tal10 を５’プライマーとして使用し、そしてTal11 を３’プライマーとして用いそしてＡＭＧをコードするゲノム配列を鋳型として使用した。エキソン５：Tal12 を５’プライマーとして使用し、そしてTal4を３’プライマーとして使用し、そしてＡＭＧをコードするゲノム配列を鋳型として使用した。
【０１０８】
最後のＰＣＲ反応を行って、上記五つのエキソンを、完全なコード配列を含有する配列に結合させた。このＰＣＲ反応において、最初のＰＣＲ反応由来の前記五つのフラグメントを鋳型として使用し、そしてTal1を５’プライマーとして用い、そしてTal4を３’プライマーとして使用した。
コード領域を含有するこの最後のＰＣＲフラグメントを用いて、（コード領域を取出し同時に各末端に約２０bpのオーバーラップ部分をつくり、組み換え事象を可能にするため）制限酵素ＳｍａＩ（またはBamHI)およびXbaIで切断したpJS026で、酵母内にて生体内組換えを行った。
【０１０９】
Tal 1 : 5'-CAA TAT AAA CGA CGG TAC CCG GGA GAT CTC CAC CATG GCG TCC CTC GTT G-3'（配列番号：２３）；
Tal 4 : 5'-CTA ATT ACA TCA TGC GGC CCT CTA GAT CAC TGC CAA CTA TCG TC-3'（配列番号：２４）；
Tal 5 : 5'-AAT TTG GGT CGC TCC TGC TCG-3'(配列番号：２５）；
Tal 6 : 5'-CGA GCA GGA GCG ACC CAA ATT ATT TCT ACT CCT GGA CAC G-3'(配列番号：２６）；
【０１１０】
Tal 7 : 5'-GAT GAG ATA GTT CGC ATA CG-3'（配列番号：２７）；
Tal 8 : 5'-CGT ATG CGA ACT ATC TCA TCG ACA ACG GCG AGG CTT CGA CTG C-3'(配列番号：２８）；
Tal 9 : 5'-CGA AGG TGG ATG AGT TCC AG-3'（配列番号：２９）；
Tal 10 : 5'-CTG GAA CTC ATC CAC CTT CGA CCT CTG GGA AGA AGT AGA AGG-3'（配列番号：３０）；
Tal 11 : 5'-GAC AAT ACT CAG ATA TCC ATC-3'（配列番号：３１）；
Tal 12 : 5'-GAT GGA TAT CTG AGT ATT GTC GAG AAA TAT ACT CCC TCA GAC G-3'（配列番号：３２）
【０１１１】
実施例８．タラロミセス・エメルソニイのグルコアミラーゼの酵母内での発現
酵母のサッカロミセス・セレビシエYNG318内で、タラロミセス・エメルソニイのＡＭＧを発現させるため、酵母発現ベクターpJS026を、上記“材料と方法”の項に記載したようにして構築した。
【０１１２】
標準の方法(Sambrooksら、“Molecular Cloning : A Laboratory Manual ”第２版1989年、コールド・スプリング・ハーバー参照）によって、タラロミセスＡＭＧをコードするＤＮＡ配列を含有するｐＪＳ０２６で、前記酵母を形質転換した。
得られた酵母細胞を、Sc-ura培地内で、３０℃にて３日間増殖させ、続いて、ＹＰＤ内で３日間増殖させた。次に、その培養物を遠心分離し、次いで、上澄み液を使用して、“材料と方法”の項に記載した熱安定性の検定を行った。
【０１１３】
酵母内で発現させたタラロミセスＡＭＧの、６８℃における熱安定性
酵母（サッカロミセス・セレビシエYNG318）内で発現させたタラロミセス・エメルソニイＡＭＧの醗酵ブロスを使用し、“熱安定性IIの測定”の項で先に述べた方法を利用して６８℃における熱安定性を測定した。試験結果を図１２に示す。
【０１１４】
実施例９．エイ・ニガーＨｏｗＢ１１２を用いて産生させた組換えタラロミセス
ＡＭＧの精製
タラロミセス・エメルソニイ遺伝子を保持するエイ・ニガーHowB112 を醗酵させて得た培養ブロス２００mlを、9000rpm で遠心分離し、次に、２０mM NaOAc （pH５）に対して一夜透析した。次にその溶液を、２０mM NaOAc 溶液（pH５）内で予め平衡させておいたＳセファロースカラム（２００ml）に注入した。グルコアミラーゼを溶出液中に集め、次に20mM NaOAc (pH4.5)内で予め平衡させておいたＱセファロースカラム（５０ml）に注入した。未結合物質をＱセファロースカラムから洗い落とし、次に２０mM ＮａＯＡｃ中０〜0.3M NaCl の直線勾配液の１０カラム容積を超える量を使用して、グルコアミラーゼを溶離した。
【０１１５】
グルコアミラーゼの画分の純度をSDS-PAGEでチェックしたところ一つのシングルバンドだけが見られた。その分子量は、野生型グルコアミラーゼについて見られるように、やはり、約７０kdalであることが見出された。マルトースに対する比活性を測定したところ、野生型酵素についてのデータによって、８．０AGU ／mg（３７℃）と２１．０AGU ／mg（６０℃）という比活性が見出された。
【０１１６】
実施例１０．動力学的パラメータ
アスペルギルス・ニガー内で発現されたアスペルギルス・ニガーのＡＭＧおよび組換えタラロミセス・エメルソニイＡＭＧによるマルトースおよびイソマルトースの加水分解反応の動力学的パラメータ。
【０１１７】
【表３】

【０１１８】
【表４】

【０１１９】
実施例１１．エイ・ニガー内で産生された組換えタラロミセス・エメルソニイＡ
ＭＧの糖化性能
タラロミセス・エメルソニイのグルコアミラーゼの糖化性能を、酸性α−アミラーゼとプルラナーゼの添加ありとなしで、各種温度で試験した。糖化反応は下記の条件下で行った。
基質：１０DEマルトデキストリン、約３０％DS（ｗ／ｗ）
温度：６０，６５または７０℃
初期pH：４．５
【０１２０】
酵素添加量：
エイ・ニガー内で産生される組換えタラロミセス・エメルソニイのグルコアミラーゼ：０．２４または0.32AGU/g DS
エイ・ニガー由来の酸性α−アミラーゼ：0.020AFAU/g DS
バシラス属細菌由来のプルラナーゼ：0.03PUN/g DS
タラロミセスＡＭＧだけを使用する場合、高添加量（0.32AGU/g DS）で添加し、その外の場合は低添加量すなわち0.24AGU/g DSで添加した。
【０１２１】
糖化
糖化に使用する基質を、マルトデキストリン（普通トウモロコシから調製した）を沸騰Milli-Q 水に溶解し、その乾燥物質を約３０％（ｗ／ｗ）に調節することによって製造した。pHは４．５に調節した（６０℃で測定）。１５０ｇの乾燥固形分に相当する基質の部分を、５００mlのブルーキャップガラスフラスコに移し、撹拌しながら、それぞれの温度の湯浴中に入れた。酵素を添加し、次に、必要に応じてpHを再調節した（インキュベーション温度において測定）。試料を定期的に採取し、HPLCで分析して、炭水化物の組成を測定した。
【０１２２】
糖化中に産生されたグルコースを下記の表に示し、左側三つの欄はグルコアミラーゼと酸性αアミラーゼとプルラナーゼによる糖化を示し、右側三つの欄はグルコアミラーゼだけによる糖化を示す。数字はDSに対する％ＤＰ１である。
【０１２３】
【表５】

【０１２４】
0.03mg/g DS に相当する0.24AGU/g DSの酵素添加量を用いて、９５％を超えるグルコースの収率を、７２hr後に得た。エイ・ニガーのＡＭＧの一般的な添加量は、0.09mg/g DS に相当する0.18AGU/g DSであり、この場合、９５〜９６％の収率でグルコースが得られる。したがって糖化法で必要な、タラロミセスＡＭＧのmg酵素タンパク質の酵素添加量は、ティー・エメルソニイＡＭＧの比活性が高いため、エイ・ニガーＡＭＧと比べて有意に低い。
【０１２５】
実施例１２．酵母内で発現された組換えタラロミセス・エメルソニイのＡＭＧの
温度安定性−Ｔ ¹ / ₂ （半減期）
酵母内で発現された組換えタラロミセス・エメルソニイのグルコアミラーゼ（実施例９に記載の方法を利用して精製）の熱安定性を、“材料と方法”の項に“ＡＭＧの熱安定性Ｉ〔Ｔ¹/₂ （半減期）〕の測定”として先に記載した方法を利用して、７０℃、pH４．５、０．２AGU ／mlで測定した。
図１３は試験結果を示す。酵母内で発現された組換えタラロミセス・エメルソニイのグルコアミラーゼのＴ¹/₂ は、測定の結果、７０℃で約１１０分であった。
【図面の簡単な説明】
【図１】 図１は、本発明の精製されたタラロミセス・エメルソニイCBS 793.97のグルコアミラーゼの分子量（Mw）を測定するのに使用したSDS-PAGEゲル（クーマシー・ブルーで染色）を示す。
１：標準マーカー
２：Ｑセファロースのプール（１．ラン）
３：Ｓセファロースのプール
【図２】 図２は、６０℃の０．５％マルトース内でのタラロミセス・エメルソニイとアスペルギルス・ニガーのグルコアミラーゼ（ＡＭＧ）のpH−活性のグラフを示す。
【図３】 図３は、タラロミセス・エメルソニイCBS 793.97のグルコアミラーゼ対アスペルギルス・ニガーのグルコアミラーゼ（ＡＭＧ）の温度−活性のグラフを示す。
【図４】 図４は、タラロミセス・エメルソニイCBS 793.97のグルコアミラーゼ対アスペルギルス・ニガーのグルコアミラーゼ（ＡＭＧ）の50mM NaOAc、0.2AGU／ml、pH4.5 における７０℃でのＴ¹/₂ （半減期）を測定するための曲線を示す。
【図５】 図５は、タラロミセス・エメルソニイのＡＭＧの遺伝子座の配列を示す。予測アミノ酸配列をヌクレオチド配列の下に示してある。四つのイントロンは小文字で示してある。コンセンサスイントロンの配列には下線を施してある。推定シグナルとプロペプチドにはそれぞれ２本の下線と点線の下線をつけてある。
【図６】 図６は、エイ・ニガーのＡＭＧ（An amg-1. pro) 、エイ・オリゼのＡＭＧ（Ao AMG. pro) 、およびタラロミセス・エメルソニイのＡＭＧ（Tal-AMG. pro) のアミノ酸配列の配列状態／比較を示す。同じアミノ酸は＊印で示してある。シグナルペプチドとプロペプチドにはそれぞれ、一本下線と二本下線を施してある。
【図７】 図７は、実施例５で使用したアスペルギルス属真菌の発現カセットｐＣａＨｊ４８３を示す。
【図８】 図８は、タラロミセス・エメルソニイのＡＭＧの遺伝子に用いるアスペルギルス属真菌の発現プラスミドpJa1518 を示す。
【図９】 図９は、エイ・ニガーの分断プラスミドの構築を示す。
【図１０】 図１０は、本発明のタラロミセス・エメルソニイのグルコアミラーゼを発現する２種の形質転換体のJaL228#5.77 とHowB112#8.10のSDS Pageゲルを示す。JaL228とHowB112 は形質転換されていない親の株である。MwはProgmaのタンパク質の分子量である。
【図１１】 図１１は、菌株Ａ．ニガーHowB112 が産生するティー・エメルソニイのAMG の熱安定性〔50mM NaOAc (pH4.5)、70℃、0.2AGU／mlで測定〕を示す。測定した結果、Ｔ¹/₂ は２０分間であった。
【図１２】 図１２は、酵母内で産生され、酵母内で発現されたティー・エメルソニイのＡＭＧおよびエイ・ニガーのＡＭＧの醗酵ブロスの６８℃における熱安定を比較する図である。
【図１３】 図１３は、酵母中で産生された組換えタラロミセス・エメルソニイＡＭＧの（７０℃、pH４．５、０．２AGU ／mlにおける熱安定性の測定試験結果を示す。測定した結果、Ｔ¹/₂ は約１１０℃であった。
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a heat-stable glucoamylase suitable for performing a starch conversion reaction that produces glucose from starch, for example. The invention also relates to the use of the thermostable glucoamylase in various methods, in particular in the saccharification step in the starch conversion process.
[0002]
Background of the Invention
Glucoamylases (1,4-α-D-glucan glucohydrase EC 3.2.1.3) are enzymes that catalyze the reaction of releasing D-glucose from non-reducing ends of starch or related oligosaccharide molecules or polysaccharide molecules. It is.
Glucoamylases are produced by a variety of filamentous fungi and yeasts, including Aspergillus niger and Aspergillus awamori.
[0003]
These glucoamylases are used commercially to convert corn starch that has already been partially hydrolyzed to glucose by α-amylase. The glucose can be further converted by glucose isomerase into a mixture of approximately the same amount of glucose and fructose. This mixture, or a further enrichment of this mixture with fructose, is a high fructose corn syrup that is commercially used and commonly used throughout the world. This syrup is the largest product in the world in production tonnage among the products produced by the enzymatic method. Among the most important industrial enzymes produced are the three enzymes involved in the conversion of starch to fructose.
[0004]
One of the major problems associated with the commercial use of glucoamylase in producing high fructose corn syrup is, for example, the commercially available Aspergillus niger glucoamylase (ie Novo Nordisk A / S is AMG). The thermal stability of glucoamylases such as those sold as) is relatively low. Commercial Aspergillus glucoamylases are not as thermally stable as α-amylase or glucose isomerase and are most active and stable at low pH compared to α-amylase or glucose isomerase. Therefore, this glucoamylase must be used in a separate container at low temperature and low pH.
[0005]
US Pat. No. 4,247,637 describes a thermostable glucoamylase having a molecular weight of about 31,000 Da derived from Talaromyces duponti suitable for saccharifying liquefied starch solutions into syrups. The glucoamylase is said to retain at least about 90% of its initial glucoamylase activity when held at 70 ° C. for 10 minutes at pH 4.5.
[0006]
US Pat. No. 4,587,215 discloses a thermostable amyloglucosidase having a molecular weight of about 45,000 Da derived from a species of Talaromyces thermophilus. This disclosed amyloglucosidase (ie, glucoamylase) loses its enzymatic activity in two distinct phases. That is, there is a period in which it declines rapidly early and then slowly declines. At 70 ° C. (pH = 5.0), the half-life upon rapid decay is about 18 minutes, and in the second phase of decay, there is no measurable loss of activity within 1 hr.
[0007]
Bunni L. et al., Enzyme Microb. Technol., 11: 370-375, 1989, from at least one form of α-amylase and one form of α-glucosidase produced by Talaromyces emersonii CBS 814.70. The literature on the production, isolation and partial characterization of the resulting extracellular starch hydrolysis system. Only α-amylase has been isolated and purified and then characterized.
[0008]
Summary of invention
The present invention is based, for example, on the discovery of a novel thermostable glucoamylase suitable for use in the saccharification step in starch conversion methods.
The terms “glucoamylase” and “AMG” are used interchangeably hereinafter.
The thermostability of the glucoamylase of the present invention can be measured using the method described in the “Materials and Methods” section below.¹/₂ Measured as (half-life).
[0009]
The inventors of the present invention have isolated, purified and characterized the thermostable glucoamylase from a strain of Talaromyces emersonii currently deposited at the Centraal bureau voor Schimmel cultures under the number CBS 793.97.
The term “isolated” when used with a protein indicates that the protein is found under conditions other than its native environment. A preferred form of isolated protein is substantially free of other proteins, particularly other homologous proteins [ie, “homologous impurities” (see below)].
[0010]
It is preferred to provide a form of the protein with a purity greater than 40%, and more preferred to provide a form of the protein with a purity greater than 60%. It is preferable to provide highly purified proteins, ie proteins with a purity greater than 80%, as measured by SDS-PAGE, more preferred is a protein with a purity greater than 95%, and even more preferred is purity. Is a protein greater than 99%.
[0011]
The term “isolated enzyme” can alternatively be referred to as “purified enzyme”.
The term “homologous impurity” refers to any impurity (eg, a polypeptide other than a polypeptide of the present invention) produced by an allogeneic cell from which the polypeptide of the present invention is first obtained.
[0012]
The isolated glucoamylase of the present invention is much more thermally stable than glucoamylases of the prior art such as Aspergillus niger glucoamylase (trade name AMG glucoamylase available from Novo Nordisk A / S). Very expensive. That T¹/₂ As a result of measuring (half-life), it was about 120 minutes at 70 ° C. (pH 4.5) as described in Example 2 below. Recombinant T. coli expressed in yeast. Emmersony AMG T¹/₂ As a result of measurement, as described in Example 12, it was about 110 minutes.
[0013]
Accordingly, in a first aspect, the present invention provides T¹/₂ It relates to an isolated enzyme having a glucoamylase activity (half-life) of at least 100 minutes at 50 mM NaOAc, 0.2 AGU / ml, pH 4.5, 70 ° C.
In the second aspect, the present invention relates to an enzyme having one or more partial sequences shown in SEQ ID NOs: 1 to 6 and having glucoamylase activity, a full-length enzyme shown in SEQ ID NO: 7, or substantially the same as these enzymes. And an enzyme having glucoamylase activity.
[0014]
The term “partial sequence” means a sequence of a partial polypeptide contained within a longer polypeptide sequence, the longer polypeptide sequence having the activity of interest.
The present invention also relates to a cloned DNA sequence encoding the glucoamylase of the present invention.
Furthermore, the present invention is a method for converting starch or partially hydrolyzed starch into a syrup containing eg dextrose, wherein the starch hydrolyzate is saccharified in the presence of the glucoamylase of the present invention. It relates to a method comprising steps.
[0015]
The objective of this invention is providing the saccharification method of the liquefied starch solution which implements the saccharification by an enzyme using the glucoamylase of this invention.
Furthermore, the invention relates to the use of the glucoamylase according to the invention in a starch conversion process, for example in a continuous starch conversion process. An embodiment of the continuous starch conversion process includes a continuous saccharification step.
The glucoamylase of the present invention can also be used in a method for producing oligosaccharides or special syrup.
Finally, the present invention relates to an isolated pure culture of the microorganism Tallalomyces emersonii CBS 793.97 or a variant thereof capable of producing the glucoamylase of the present invention.
[0016]
Detailed Disclosure of the Invention
The present invention is based, for example, on the discovery of a novel thermostable glucoamylase suitable for use in the saccharification step of the starch conversion process.
The inventors of the present invention isolated, purified and characterized the glucoamylase from the strain of Talalomyces emersonii CBS 793.97. It was revealed that this glucoamylase has a very high thermal stability as compared with the conventional glucoamylase.
[0017]
Thus, in a first aspect, the present invention relates to Tm at 50 mM NaOAc, 0.2 AGU / ml, pH 4.5, 70 ° C.¹/₂ It relates to an isolated enzyme having glucoamylase activity with a (half-life) of at least 100 minutes, for example 100-140 minutes.
Isolated Talaromyces emersonii CBS 793.97 glucoamylase T¹/₂ The (half-life) was measured to be about 120 minutes at 70 ° C. as described in Example 2 below, and as described in Example 12, tea-emersony produced in yeast. Was about 110 minutes.
[0018]
As a result of SDS-PAGE measurement, the molecular weight of the isolated glucoamylase was found to be about 70 KDa. Furthermore, the PI of the enzyme was less than 3.5 as a result of measurement using isoelectric focusing.
The isoelectric point PI is the value obtained when the enzyme molecule complex (optionally bound with ions such as metals) is neutral, that is, the total electrostatic charge of the complex (the net electrostatic charge, NEC] is defined as the pH value when equal to zero. Of course, this summation must take into account whether the electrostatic charge is positive or negative.
Substantially homologous enzymes with the same advantageous properties are obtained from other microorganisms, in particular fungal organisms such as filamentous fungi, more particularly from other strains of the fungus of the genus Talalomyces, in particular from other strains of Talalomyces emersonii It is expected that
[0019]
Deposited microorganism
An isolate of a filamentous fungal strain from which the glucoamylase of the present invention has been isolated has been deposited with the Centraal bureau voor Schimmel cultures in Baan AG 3740 PO Box 273, the Netherlands, for patent procedures dated below. CBS, the international depositary authority under the Budapest Treaty, will store microorganisms deposited in accordance with the ninth regulation of the Convention.
Deposit date: June 2, 1997
Depositor's reference number: NN049253
Name of CBS: CBS 793.97

[0020]
The isolate of the filamentous fungus Talalomyces emersonii CBS No. 793.97 is pending a patent pending by the Director of the Patent and Trademark Office while the patent application is pending, as determined by CFR §1.14 and 35U.SC Deposited on condition that an isolated fungus is available. The deposited microorganism is a substantially pure culture of a fungal isolate. The deposited microorganism can be obtained when requested by the foreign patent law of the country in which the corresponding application of the application or the successor application of the application was filed. However, it should be understood that the use of deposited microorganisms does not constitute a license for carrying out the invention, deviating from the patent right granted by the government's decision.
[0021]
Amino acid sequence of Talalomyces emersonii glucoamylase
The inventors of the present invention determined the sequence of a thermostable glucoamylase from Talalomyces emersonii CBS 793.97. This is described in Example 3 below. According to the present invention, the A. thalamomyces AMG has a substitution of Asp145Asn (ie D145N) (using the numbering of SEQ ID NO: 7).
[0022]
Accordingly, the present invention provides an isolated enzyme having glucoamylase activity comprising one or more of the partial sequences shown in SEQ ID NOs: 1 to 6 or the full-length sequence shown in SEQ ID NO: 7, or substantially the enzyme. The present invention relates to an enzyme having homologous glucoamylase activity. SEQ ID NO: 34 shows the full-length sequence including the signal peptide of amino acids No. 1-27 and the prepro peptide.
[0023]
Protein sequence homology
Homology between two glucoamylases is measured as the degree of identity of the two protein sequences, indicating the derivation of the first sequence from the second sequence. The homology is the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computers Group, USA 53711, Madison, Wisconsin, Science Drive 575) and other technical fields such as gap. (Needleman, SB and Wunsch, CD, Journal of Molecular Biology, 48, 443-453, 1970). To compare polypeptide sequences, the following settings are used: gap, gap generation penalty 3.0 and gap extension penalty 0.1.
[0024]
According to the present invention, the “substantially homologous” amino acid sequence is preferably at least 80%, at least 90%, more preferably at least 95 with the partial amino acid sequence shown in SEQ ID NO: 1-6 or SEQ ID NO: 7. %, More preferably 97%, and most preferably at least 99% identity.
[0025]
The cloned DNA sequence of Talalomyces emersonii
The present invention also relates to a cloned DNA sequence encoding the enzyme of the present invention exhibiting glucoamylase activity,
(A) a portion encoding the glucoamylase of the DNA sequence shown in SEQ ID NO: 33;
(B) a DNA sequence represented by positions 649 to 2724 of SEQ ID NO: 33 or a complementary strand thereof;
(C) an analog of said DNA sequence that is at least 80% homologous to the DNA sequence defined in (a) or (b) above;
[0026]
(D) a DNA sequence that forms a hybrid with a double-stranded DNA probe comprising the sequence shown in SEQ ID NO: 33 at 649 to 2724 with low stringency;
(E) a sequence that does not form a hybrid with the sequence of (b) because the genetic code is degenerate, or (f) however, an amino acid sequence that is exactly the same as a polypeptide encoded by any one of these DNA sequences A DNA sequence encoding a polypeptide having: or
(G) a DNA sequence that is a fragment of the DNA sequence specified in (a), (b), (c), (d) or (e) above;
Contains.
[0027]
The mature part of the AMG of the present invention is encoded by the DNA sequence at positions 728 to 2724 of SEQ ID NO: 33. When expressing the AMG of the present invention in yeast such as Saccharomyces cerevisiae YNG318, the intron needs to be cleaved as described in Example 7.
[0028]
DNA sequence homology
The homology of the above DNA sequences is determined as the degree of identity between two sequences indicating that the first sequence is derived from the second sequence. The homology is known in the art from computer programs such as GAP (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, USA, 53711, Wisconsin, GCG program package). Madison, Science Drive 575), which can be determined appropriately (Needleman, SB and Wunsch, CD, Journal of Molecular Biology, 48, 443-453, 1970).
[0029]
When GAP with a GAP generation penalty of 5.0 and a GAP extension penalty of 0.3 is used to compare the DNA sequences, the coding region of the above similar DNA sequence is the AMG of the DNA sequence shown in SEQ ID NO: 33. Preferably at least 80%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with the region coding for or the portion coding for glucoamylase with or without introns .
[0030]
Hybrid formation
The sequence shown at positions 649-2748 of SEQ ID NO: 33 (ie, the portion encoding AMG), at least under low stringency conditions, but preferably under moderate stringency conditions or high stringency conditions The hybridization conditions for defining the similar DNA sequence defined in d) above to form a hybrid with a double-stranded DNA probe containing, will be described in detail below.
[0031]
Suitable test conditions to confirm low, moderate or high stringency hybridization between the nucleotide probe and the sequence of homologous DNA or RNA include DNA fragments or RNA that cause hybridization. Pre-soak for 10 minutes in 5 × SSC (sodium chloride / sodium citrate, Sambrook et al., 1989) containing 5 × SSC, 5 × Denhardt's solution (Sambrook et al., 1989), 0.5 Prehybridization of the filter in a solution of% SDS and 100 μg / ml sonicated denatured salmon sperm DNA (Sambrook et al., 1989); then randomly primed (Feinberg, AP and Vogelstein B., Anal. Biochem., 132, 6-13, 1983),³²Labeled with P-dCTP (specific activity> 1 × 10⁹Cpm / μg) is included in the same solution containing the probe, 12 hrs hybridization at about 45 ° C.
[0032]
The filter is then placed in 2 × SSC, 0.5% SDS for 30 minutes at about 55 ° C. (low stringency), more preferably at about 60 ° C. (medium stringency), even more preferred. At about 65 ° C. (medium / high stringency), even more preferably at about 70 ° C. (high stringency) and even more preferably at about 75 ° C. (very high stringency), twice Wash.
Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using X-ray film.
[0033]
Starch conversion
The present invention provides a method for producing glucose and the like from starch using the thermostable glucoamylase of the present invention. This method generally involves partially hydrolyzing the precursor starch in the presence of α-amylase and then the α- (1 → 4) and α- (1 → 6) glucosides in the presence of glucoamylase. It further comprises hydrolyzing by cleaving the bond to release D-glucose from the non-reducing end of the starch or to release related oligosaccharide molecules and polysaccharide molecules.
[0034]
The partial hydrolysis reaction of precursor starch using α-amylase performs initial decomposition of starch molecules by hydrolyzing internal α- (1 → 4) bonds. In commercial applications, the initial hydrolysis reaction using α-amylase is performed at a temperature of about 105 ° C. Typically, very high starch concentrations with a solids content of 30-40% are processed. The initial hydrolysis is usually performed at the high temperature for 5 minutes. The partially hydrolyzed starch is then transferred to a second tank and incubated for about 1 hr at a temperature of 85-90 ° C. to obtain a dextrose equivalent (DE) of 10-15.
[0035]
The step of further hydrolysis in the presence of glucoamylase to release D-glucose from the non-reducing end of the starch or to release related oligosaccharide and polysaccharide molecules is usually performed in separate tanks. It is performed at a low temperature of 30-60 ° C. The temperature of the substrate solution is preferably lowered to 55-60 ° C. The pH of the solution is lowered from 6-6.5 to the range of 3-5.5. The pH of the solution is preferably 4 to 4.5. Glucoamylase is added to the solution and the reaction is carried out for 24-72 hours, preferably 36-48 hours.
[0036]
By using the thermostable glucoamylase of the present invention, the saccharification method can be carried out at a higher temperature than the conventional batch saccharification method. According to the present invention, saccharification can be carried out at a temperature in the range of 60 ° C. to 80 ° C., preferably 63-75 ° C. This is true for both conventional batch processes (above) and continuous saccharification processes.
[0037]
Indeed, continuous saccharification processes involving one or more membrane separation or filtration steps must be performed at temperatures above 60 ° C. so that a reasonably high flux can be maintained on the membrane. Therefore, the thermostable glucoamylase of the present invention enables a large-scale continuous saccharification method to be carried out within a reasonable price and in a time acceptable as an industrial saccharification method. According to the present invention, the saccharification time can be further shortened.
[0038]
The activity of the glucoamylase of the present invention is generally considerably higher at a temperature of 60 ° C. to 80 ° C. than the conventionally used temperature of 30 to 60 ° C. Therefore, by increasing the temperature at which glucoamylase acts, the saccharification method can be carried out within a shorter time or can be carried out with a reduced amount of enzyme.
[0039]
The heat stability of the glucoamylase of the present invention is very high compared to, for example, the commercially available Aspergillus niger glucoamylase (ie, AMG), so it must be added to replace the glucoamylase that is inactivated during the saccharification process. There is a reduction in the amount of glucoamylase. According to the present invention, more glucoamylase is maintained in the active state during the saccharification process. Furthermore, if the saccharification step is carried out at a temperature higher than 63 ° C., the risk of contamination by microorganisms is also reduced.
[0040]
By using a glucoamylase having a high specific activity (measured as activity against maltose), the amount of enzyme required in the saccharification step can be reduced.
Examples of the saccharification step that can advantageously use the glucoamylase of the present invention include the steps described in JP3-224493, JP1-191693, JP62-272987, and EP452,238.
In another aspect, the present invention relates to a method for saccharifying a liquefied starch solution comprising an enzymatic saccharification step using the glucoamylase of the present invention.
[0041]
The glucoamylase of the present invention can be used in the method of the present invention in combination with an enzyme that hydrolyzes only the α- (1 → 6) glucoside bond of a molecule having at least four glucosyl residues. The glucoamylase of the present invention is preferably used in combination with pullulanase or isoamylase. For the use of isoamylases and pullulanases to perform debranching, the molecular properties of these enzymes and the potential use of these enzymes and glucoamylases, see GMAVan Beynum et al., “Starch Conversion Technology”, Marcel. Dekker New York, 1985, pages 101-142.
[0042]
In another aspect, the invention relates to the use of the glucoamylase of the invention in a starch conversion process.
Furthermore, the glucoamylase of the present invention can be used in a continuous starch conversion process including a continuous saccharification step.
[0043]
The glucoamylase of the present invention can be used in an immobilized form. It is suitable and often used to produce special syrups such as maltose syrup, and also to obtain a raffinate stream of oligosaccharides in connection with the production of fructose syrup.
The glucoamylase of the present invention can also be used in a method for producing ethanol for fuels or beverages, or in a fermentation method for producing organic compounds such as citric acid, ascorbic acid, lysine, and glutamic acid. Can be used.
[0044]
Materials and methods
material
Enzymes:
The deposited glucoamylase from the filamentous fungus Talaromyces emersonii CBS No. 793.97 was deposited in the PO Box 273 of the Centraal bureau voor Schimmel cultures, Bern, 3740AG for patent proceedings on the following date. CBS is an international depositary authority under the Budapest Treaty and stores deposited microorganisms in accordance with the ninth rule of the treaty.
Deposit date: June 2, 1997
Depositor's reference number: NN049253
Name of CBS: CBS 793.97

[0045]
A glucoamylase G1 derived from Aspergillus niger disclosed in Boel et al., EMBO J., Volume 3 (5) Nos. 1097 to 1102, 1984, is available from Novo Nordisk and is shown in SEQ ID NO: 9. It is.
[0046]
Strains:
JaL228: The construction of this strain is described in International Patent Application Publication No. WO 98/12300. SM0110: The construction of this strain is described in Example 6.
Yeast strain: Saccharomyces cerevisiae YNG318: MATa leu2-D2 Ura3-52 his4-539 pep4-D1 [Ci1 +].
[0047]
Genes:
The gene for A. niger's G1 glucoamylase is shown in SEQ ID NO: 8. The intron-containing gene of T. melsonii glucoamylase is shown in FIG. 5 and SEQ ID NO: 33. These introns are shown in FIG.
[0048]
Plasmids
pJS0026 (S. cerevisiae expression plasmid) (JSOkkels, 1996 “A URA3-promoter deletion in a pYES vector increases the expression level of a fungal lipase in Saccharomyces cerevisiae. Recombinant DNA Biotechnology III: The Integration of Biological and Engineering Sciences, vol.782 of the Annals of the New York Academy of Sciences) More specifically, the inducible GAL1-promoter of pYES2.0 is a constitutively expressed TPI (triose phosphate isomerase) from Saccharomyces cerevisiae. -Expression plasmid pJS026 is derived from pYES2.0 by substitution with a promoter (Albert and Karwasaki, J. Mol. Appl Genet., Pp. 419-434, 1982) and then deletion of the URA3 promoter part .
[0049]
pJaL497; the construction of this plasmid is described in Example 5.
pJaL507; the construction of this plasmid is described in Example 5.
pJaL510; the construction of this plasmid is described in Example 5.
pJaL511; the construction of this plasmid is described in Example 5.
pJaL518; the construction of this plasmid is described in Example 6.
pCaHj483; the construction of this plasmid is described in Example 6.
pJRoy10; the construction of this plasmid is described in Example 6.
pJRoy17; the construction of this plasmid is described in Example 6.
pSM0127; construction of this plasmid is described in Example 6.
pCR ™ II; Invitrogen Corpora, San Diego, California, USA
Available from tion.
[0050]
apparatus:
Automated DNA sequence analyzer (Applied Biosystems Model 377)
[0051]
Culture medium:
SC-ura medium:
Yeast Nitrogen w / o ami 7.5g
11.3 g of succinic acid (Ravsyre)
6.8 g of NaOH
Casamino acid w / o bit 5.6g
Tryptophan 0.1g
Distilled water (remaining amount) 1000ml
Autoclave at 121 ° C. for 20 minutes.
4 ml from a sterile stock solution of 5% threonine is added to a volume of 900 ml along with 100 ml of sterile 20% glucose.
[0052]
YPD medium:
Yeast extract 10g
Peptone 20g
Distilled water (remaining amount) 1000ml
Autoclave at 121 ° C. for 20 minutes.
Add 100 ml of sterile 20% glucose to 900 ml.
[0053]
Method:
Measurement of AGU activity
One unit of Novo amyloglucosidase unit (AGU) is defined as the amount of enzyme that hydrolyzes 1 micromole of maltose per minute under the following standard conditions.
Substrate ... Maltose
Temperature ... 25 ° C
pH …… 4.3 (acetate buffer)
Reaction time ... 30 minutes
Detailed instructions for this analysis method (AF22) are available upon request.
[0054]
Measurement of PUN activity
PUN is defined as the amount of enzyme that hydrolyzes pullulan (0.2% pullulan, 40 ° C., pH 5.0) to release reducing carbohydrate per minute with a reducing power equal to 1 micromole of glucose To do.
AFAU Activity measurement
The activity of AFAU is calculated and calculated as the reducing power of starch at a concentration of 0.17 g / l at pH 2.5 and 40 ° C., and measured by iodine-starch reaction.
[0055]
Thermal stability of AMG I [T ¹ / ₂ (Half-life)]
Thermostability of glucoamylase [T¹/₂ (Measured as half-life)] is tested using the following method. 950 μl of 50 mM sodium acetate buffer (pH 4.5) (NaOAc) is incubated at 70 ° C. for 5 minutes. Add 50 μl enzyme (4 AGU / ml) in buffer. Two samples of 40 μl are taken over 0-360 minutes at fixed time intervals and cooled on ice. After cooling these samples, the residual enzyme activity is measured using the AGU assay (described above).
The activity (AGU / ml) measured before incubation (0 min) is used as a reference (100%). T¹/₂ Is the time for the specific activity percentage to drop to 50%.
[0056]
Thermal stability II Measurement
Mix 1600 μl supernatant with 400 μl 0.5 M NaAC (pH 4.5).
Each of the seven Eppendorf tubes containing 250 μl of the above mixture is incubated on a Perkin Elmer thermocycler at 68 ° C. or 70 ° C. for 0, 5, 10, 20, 30, 45 and 60 minutes.
[0057]
100 μl from each mixture is mixed with 100 μl of 5 mM CNPG3 (2-chloro-4-nitrophenyl-α-maltotrioside from genzyme) in a microtiter well. After incubating at 37 ° C. for 30 minutes, the absorbance at 405 nm is measured.
[0058]
Measurement of specific activity of glucoamylase
750 μl of substrate is incubated for 5 minutes at the selected temperature, eg 37 ° C., 60 ° C. or 70 ° C.
50 μl of enzyme diluted with sodium acetate was added and the activity was measured using the above AGU standard method. By adding 50 μl of enzyme diluted in sodium acetate to 750 μl of preheated substrate, the dynamic parameter k_cat And K_m Is measured at 45 ° C. 100 μl aliquots are removed after 0, 3, 6, 9 and 12 minutes and transferred to 100 μl 0.4 M sodium hydroxide to stop the reaction. Take one blank.
[0059]
Transfer 20 μl to a microtiter plate and add 200 μl GOD-Perid solution. After incubation at room temperature for 30 minutes, the absorbance at 650 nm is measured. Using glucose as a reference, the specific activity is k_cat (Sec^-1).
[0060]
Aspergillus oryzae ( Aspergillus oryzae ) Transformation (general method)
100 ml of YPD (Sherman et al. “Methods in Yeast Genetics”, 1981, Cold Spring Harbor Laboratory) is inoculated with A. oryzae spores and then incubated for 24 hours with agitation. Harvest the mycelium by filtering through miracloth and then 0.6M MaSO_Four Wash with 200ml. The mycelium is 1.2M MaSO_Four, 10mM NaHPO_Four Suspend in 15 ml of solution (pH 5.8). The suspension is cooled on ice and 1 ml of buffer containing 120 mg Novozym ™ 234 is added. After 5 minutes, 1 ml of 12 mg / ml BSA (Sigma type H25) is added and 1.5-2 at 37 ° C. with gentle agitation until a large number of protoplasts are visible to the naked eye in the sample to be examined under the microscope. Incubation continued for 5 hr.
[0061]
The resulting suspension is filtered through miracloth and the filtrate is transferred to a sterile test tube and covered with 5 ml of a solution of 0.6 M sorbitol, 100 mM Tris-HCl (pH 7.0). Centrifugation at 1000 g for 15 minutes and protoplast_Four Collect from the top of the cushion. 2 volumes of STC [1.2 M sorbitol, 10 mM Tris-HCl (pH 7.5), 10 mM CaCl₂Is added to the suspension of protoplasts and the mixture is centrifuged at 1000 g for 5 minutes. The resulting protoplast pellet is resuspended in 3 ml of STC and then pelleted again. Repeat this operation. Finally, the protoplasts are resuspended in 0.2-1 ml STC.
[0062]
100 μl of the protoplast suspension was added to 5-25 μg of p3SR2 [A. nidulans described in Hynes et al., Mol. And Cel. Biol. Vol. 3, No. 8, pages 1430-1439, August 1983. (1) plasmid containing the amds gene) and 10 μl of STC. The resulting mixture is left at room temperature for 25 minutes. 60% PEG4000 (BDH29576), 10 mM CaCl₂And 0.2 ml of a solution of 10 mM Tris HCl (pH 7.5) is added and mixed carefully (twice), and finally 0.85 ml of the same solution is added and mixed carefully. The mixture is left at room temperature for 25 minutes, centrifuged at 2,500 g for 15 minutes, and the resulting pellet is resuspended in 2 ml of 1.2 M sorbitol.
[0063]
After another precipitation, the protoplasts were added to a minimal plate (Cove, Biochem. Biophys. Acta) containing 1.0 M sucrose (pH 7.0), 10 mM acetamide (nitrogen source) and 20 mM CsCl. 113, pp. 51-56, 1966) and smear over to prevent background growth. After incubation at 37 ° C. for 4-7 days, the spores are collected, suspended in sterile water and smeared as a single colony. After repeating this procedure and performing a second re-isolation, single colony spores are stored as defined transformants.
[0064]
Fed-batch fermentation
Fed-batch fermentation is carried out in a medium containing urea and yeast extract as maltodextrin nitrogen source as carbon source. This fed-batch fermentation is performed by inoculating a shake flask culture of the fungal host cells of interest into a medium containing 3.5% of the carbon source and 0.5% of the nitrogen source. After culturing at 34 ° C. for 2 hours at pH 5.0, continuous supply of additional carbon source and nitrogen source is started.
[0065]
Keep the carbon source as a limiting factor and ensure that oxygen is present in excess. The fed-batch culture continues for 4 days, after which the enzyme can be recovered by centrifugation, ultrafiltration, clear filtration and germ filtration. Further purification can be performed by anion exchange chromatography methods known in the art.
[0066]
Transformation of Saccharomyces cerevisiae YNG318
The DNA fragment and the open vector are mixed and then transformed into the yeast Saccharomyces cerevisiae YNG318 by standard methods.
[0067]
Example
Example 1. Purification
3500 ml of 0.05 AGU / ml tea-emersony culture broth obtained by wild-type fermentation was centrifuged at 9000 rpm, then filtered under reduced pressure using filter paper, and finally blank filtration was performed. The enzyme was then purified using the following procedure.
Phenyl Sepharose (250 ml): 1.3 M AMS / 10 mM Tris / 2 mM CaCl₂ , PH 7; 10 mM Tris / 2 mM CaCl₂ Elute at pH 7.
Dialysis: 20 mM NaAc, 2 mM CaCl₂ , PH 5.
Q Sepharose (100ml): 20mM NaAc, 2mM CaCl₂ Elution with a linear gradient from 0 to 0.4 M NaCl over 10 column volumes.
Dialysis: 20 mM NaAc, 2 mM CaCl₂ , PH 5.
Color removal: 0.5% coal, 10 minutes.
Q Sepharose (20 ml): 20 mM NaAc, 2 mM CaCl₂Elute with a linear gradient of 0-0.4M NaCl over 10 column volumes, pH 4.5;
Dialysis: 20 mM, 2 mM CaCl₂ , PH 5.
S Sepharose (1 ml): 5 mM citric acid pH 2.9; eluted with a linear gradient of 0-0.3 M NaCl over 10 column volumes.
After the S Sepharose step, enzyme purity greater than 90% was obtained.
[0068]
Example 2 Characterization of Talalomyces emersonii glucoamylase
Purified Tallalomyces emersonii CBS 793.97 glucoamylase was used to characterize.
Molecular weight (M_w )
The molecular weight measured by SDS-PAGE was about 70 KDa as shown in FIG.
PI
PI was less than 3.5 as determined by isoelectric focusing (Amploline PAG, pH 3.5-9.5 available from Pharmacia).
[0069]
pH characteristics
The pH-activity dependency of Talalomyces emersonii glucoamylase was measured and compared with that of Aspergillus niger glucoamylase.
The properties of pH activity were measured at 60 ° C. in 0.1 M sodium acetate using 0.5% maltose as a substrate. Measurements were made on duplicate samples containing 0.1-1 AGU / ml. The test results are shown in FIG.
[0070]
Temperature characteristics
The temperature-activity dependency of the Talaromyces emersonii glucoamylase of the present invention was measured and compared with the properties of Aspergillus niger glucoamylase. The reaction was incubated by incubating 200 μl of 0.5% maltose (pH 4.3) at 37, 50, 60, 70, 75, 80 and 90 ° C. and then adding 10 μl of enzyme (0.25 AGU / ml). Started. The reaction time was 10 minutes. The test results are shown in FIG.
[0071]
Temperature stability -T¹/₂ (Half life)
The thermal stability of the Talalomyces emersonii glucoamylase was measured and compared with the thermal stability of the Aspergillus niger glucoamylase. The method used is described in the “Materials and Methods” section under “AMG Thermal Stability I [T¹/₂ (Half-life)] ”as described above.
When the half-life of Talalomyces emersonii glucoamylase was measured, it was about 120 minutes at 70 ° C. Aspergillus niger glucoamylase T¹/₂ Was 7 minutes as measured under the same conditions (see FIG. 4).
[0072]
Specific activity
As a result of measuring the extension coefficient based on the absorbance at 280 nm and the protein concentration, ε = 2.44 ml / mg^* cm. Next, the specific activity against maltose at 37 ° C. was calculated and found to be 7.3 AGU / mg. Since the purity of the sample was about 90%, the corrected specific activity is 8.0 AGU / mg. The following specific activities were measured.
[0073]
[Table 1]

[0074]
Example 3 FIG. The amino acid sequence of the N-terminal of T. melsonii glucoamylase
Decision
The amino acid sequence at the N-terminus of T. melsonii glucoamylase was determined by SDS-PAGE and electrophoretic transfer to a PVDF membrane. The peptide was a peptide derived from a glucoamylase that was reduced and S-carboxymethylated by cleavage with a lysyl-specific protease. The resulting peptide was fractionated and then purified again using RP-HPLC before determining the N-terminal sequence.
[0075]
N-terminal sequence (SEQ ID NO: 1):
Ala Asn Gly Ser Leu Asp Ser Phe Leu Ala Thr Glu Xaa Pro Ile Ala
Leu Gln Gly Val Leu Asn Asn Ile Gly
Peptide 1 (SEQ ID NO: 2):
Val Gln Thr Ile Ser Asn Pro Ser Gly Asp Leu Ser Thr Gly Gly Leu
Gly Glu Pro Lys
[0076]
Peptide 2 (SEQ ID NO: 3):
Xaa Asn Val Asn Glu Thr Ala Phe Thr Gly Pro Xaa Gly Arg Pro Gln
Arg Asp Gly Pro Ala Leu
Peptide 3 (SEQ ID NO: 4):
Asp Val Asn Ser Ile Leu Gly Ser Ile His Thr Phe Asp Pro Ala Gly
Gly Cys Asp Asp Ser Thr Phe Gln Pro Cys Ser Ala Arg Ala Leu Ala
Asn His Lys
[0077]
Peptide 4 (SEQ ID NO: 5):
Thr Xaa Ala Ala Ala Glu Gln Leu Tyr Asp Ala Ile Tyr Gln Trp Lys
Peptide 5 (SEQ ID NO: 6):
Ala Gln Thr Asp Gly Thr Ile Val Trp Glu Asp Asp Pro Asn Arg Ser
Tyr Thr Val Pro Ala Tyr Cys Gly Gln Thr Thr Ala Ile Leu Asp Asp
Ser Trp Gln
Xaa means a residue that could not be specified.
[0078]
Example 4 Full-length ti emersonii glucoamylase
The amino acid sequence of the full-length T. emersonii glucoamylase shown in SEQ ID NO: 7 was identified using standard methods.
[0079]
Example 5 FIG. Talaromyces emersonii glucoamylase gene clone
And sequencing
The part of the gene of Talalomyces emersonii AMG that is cloned by PCR.
In order to clone Tallaromyces emersonii AMG, the degenerate primers shown in Table 2 below were designed to amplify a portion of the AMG gene by PCR.
[0080]
[Table 2]

[0081]
Genomic DNA from Talalomyces emersonii was prepared from protoplasts made by standard methods (see, for example, Christensen et al., Biotechnology, Vol. 6, pp. 1419-1422, 1989) and used as a template for PCR reactions. Taq-polymerase 2.5 units, A. oryzae genomic DNA 100 ng, 50 mM KCl, 10 mM Tris-HCl (pH 8.0), 1.5 mM MgCl₂ , 250 nM of each dNTP, and 100 pM of each of the following primers: 10434/117360, 104341/117361, 10435/117360, 104341/117361, 104341/127420, and 10434/127420, and the amplification reaction was performed in a volume of 100 μl. .
[0082]
Amplification was performed on Perkin-Elmer Cetus DNA Termal 480 and consisted of 1 cycle at 94 ° C for 3 minutes followed by 30 cycles of 94 ° C for 1 minute, 40 ° C for 30 seconds and 72 ° C for 1 minute. Amplification. Only PCR reaction 10234/117360 provided the product. Four bands of size 1400, 800, 650 and 525 bp were detected. All four bands were purified and cloned into the vector pCR® 2.1 [Invitrogen®]. The sequence of several clones from each band was determined, and the sequence was compared with A. Niger AMG. As a result, it was revealed that the clone derived from the 650 bp band encodes the N-terminal part of Talalomyces emersonii AMG. This clone was named pJaL497.
[0083]
In order to obtain more of the gene, a specific primer (123036: 5′-GTGAGCCCAAGTTCAATGTG-3 ′ (SEQ ID NO: 15) was prepared with the sequence of clone pJaL497. The PCR fragment was cloned into the vector pCR2.1 and sequenced by sequencing, so that the clone was cleaved from the C-terminal part of Talalomyces emersonii AMG. The clone was named pJaL507.
[0084]
Genomic restriction mapping and cloning of genomic clones
The above two clones pJaL497 and pJaL507 accounted for about 95% of the AMG gene. In order to clone the missing part of the AMG gene, these two PCR fragments were probed with a single restriction enzyme or a combination of several restriction enzymes and digested with Talaromyces emersonii. Genomic restriction maps were generated by use for Southern blots of genomic DNA. This map shows that the gene for Talalomyces emersonii AMG is located in two EcoRI fragments, approximately 5.6 kb and approximately 6.3 kb, respectively.
[0085]
Talaromyces emersonii genomic DNA was digested with EcoRI and a 4-7 kb fragment was purified and used as described in the manufacturer's instructions (Stratagene) in λ2APII. A partial genome library was constructed. The library was first constructed with a 0.7 kb EcoRI fragment from pJaL497 (the N-terminal of the AMG gene).¹/₂ Was used as a probe to obtain an AMG gene start. One clone was obtained and named pJaL511. A 0.75 kb EcoRV fragment derived from pJaL507 (the C-terminal of the AMG gene)¹/₂ Was used as a probe to perform a second selection of the library to obtain the C-terminus of the AMG gene. One clone was obtained and named pJaL510.
[0086]
Sequence analysis of the gene of Talalomyces emersonii AMG
Plasmids: pJaL497, pJaL507, pJaL510 and pJaL511 and their subclones were sequenced using the standard reverse and forward primers for pUC to obtain the sequence of the AMG gene. The remaining gab was closed using specific oligonucleotides as primers.
[0087]
A potential intron was found by comparing the above sequence with the consensus sequence of introns and the sequence of AMG niger AMG in Aspergillus fungi. The nucleotide sequence of Talalomyces emersonii AMG encodes a protein of 618 amino acids and has an open reading frame interrupted by four introns of 57 bp, 55 bp, 48 bp and 59 bp, respectively. The nucleotide sequence (including intron) and the deduced amino acid sequence are shown in FIG.
[0088]
The DNA sequence (containing introns) is shown in SEQ ID NO: 33, and the sequence of Talaromyces emersonii AMG (containing signal sequences 1 to 27) is shown in SEQ ID NO: 34. When the putative amino acid sequence was compared with A. oryzae AMG and A. niger AMG, the identities were 60.1% and 60.5%, respectively. The arrangement state of the amino acid sequence shown in FIG. 6 indicates that Talaromyces AMG has a very short hinge part between the catalytic domain and the starch-binding domain. This is also seen in A-Olysee AMG.
[0089]
Example 6 Construction of Aspergillus vector pCaHj483
The construction of pCaHj483 is shown in FIG. This plasmid is constructed from the following fragments:
a) Vector pToC65 digested with EcoRI and XbaI (International Patent Application Publication No. WO91 / 17243).
[0090]
b) A 2.7 kb XbaI fragment from A. Nidulans carrying the amds gene (C. M. Corrick et al., Gene 53: 63-71 1987). This amds gene is used as a selectable marker when transforming fungi. The amds gene has been transformed to destroy the BamHI site normally present in the gene. This disruption was performed by introducing a silent point mutation using primer: 5′-AGAAATCGGGTATCCTTTCAG-3 ′ (SEQ ID NO: 16).
[0091]
c) A 0.6 kb EcoRI / BamHI fragment carrying the A. niger NA2 promoter fused to a 60 bp DNA fragment of the sequence encoding the 5'-untranslated end of the A. nidulans tpi gene mRNA. The NA2 promoter is isolated from plasmid pNA2 (described in International Patent Application Publication No. WO89 / 01969) and then fused by PCR to a 60 bp tpi sequence. Primers encoding the 60 bp tpi sequence were the following sequences:
[0092]
5'-GCTCCTCATGGTGGATCCCCAGTTGTGTATATAGAGGATTGAGGAAGGAAGAGAAGTGTGGATAGAGGTAAATTGAGTTGGAAACTCCAAGCATGGCATCCTTGC-3 '(SEQ ID NO: 17)
d) A 675 bp XbaI fragment retaining the transcription terminator of A. niger glucoamylase. This fragment was isolated from the plasmid pICAMG / Term (described in EP 0238023).
The BamHI site of fragment c was connected to the first XbaI site of the transcription terminator of fragment d via a pIC19R linker (BamHI to XbaI).
[0093]
Construction of AMG expression plasmid pJaL518
The coding region of the Talaromyces emersonii AMG gene contains the following two oligonucleotide primers:
Primer 139746: 5'-GACAGATCTCCACCATGGCGTCCCTCGTTG-3 ′ (SEQ ID NO: 18), and
Primer 139747: 5'-GACCTCGAGTCACTGCCAACTATCGTC-3 ′ (SEQ ID NO: 19)
Was amplified by PCR. The underlined region indicates the sequence present in the Talaromyces emersonii AMG gene. To facilitate cloning, a restriction enzyme site was inserted at the 5 'end of each primer. Primer 139746 contains a BglIII site and primer 139747 contains an XhoI site.
[0094]
Talaromyces emersonii genomic DNA was used as a template for the PCR reaction. The PCR reaction consisted of a reaction buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.0)) containing 2.5 units of Taq polymerase, 100 ng of pS02, 25 nM of each dNTP, and 10 pmol of each of the two primers. 1.5 mM MgCl₂ ) Performed in a volume of 100 μl.
[0095]
Amplification was performed with Perkin-Elmer Cetus DNA Termal 480, which consisted of one cycle of 94 ° C for 3 minutes followed by 25 cycles of 94 ° C for 1 minute, 55 ° C for 30 seconds and 72 ° C for 1 minute. ing. This PCR reaction produced a single DNA fragment 2099 bp in length. This fragment was digested with BglII and XhoI, then isolated by gel electrophoresis, purified and then cloned into pCaHj483 digested with BamHI and XhoI to give a plasmid, which was designated pJaL518. Thus, plasmid pJaL518 was constructed to obtain a fungal expression plasmid (FIG. 8) for the Talaromyces emersonii AMG gene.
[0096]
Construction of Aspergillus niger strain SM0110
1. Cloning of A. niger's pyrG gene
A Niger BO-1 was made in EMBL4 as described in the manufacturer's instructions. DIG-labeled oligonucleotide [PyrG: 5′-CCCTCACCAGGGGAATGCTGCAGTTGATG-3 ′ (SEQ ID NO: SEQ ID NO :) designed from the published Aspergillus niger sequence (Wilson et al., Nucleic Acids Res., 16: 2339-2339 1988) 20)] to select the library. A positive EMBL4 clone that hybridized to the DIG probe was isolated from the BO-1 library, then a 3.9 kb Xbal fragment containing the pyrG gene was subcloned from EMBL4 and then cloned into pUC118 to generate pJRoy10. Produced.
[0097]
2. Cloning of the gene for A. niger glucoamylase (AMG)
Oligonucleotide 950847: 5′-CGCCATTCTCGGCGACTT-3 ′ (SEQ ID NO: 21) and oligonucleotide 951216: 5′-CGCCGCGGTATTCTGCAG-3 ′ (SEQ ID NO: 22) [published Aspergillus niger sequence (Boel et al., A DIG-labeled PCR fragment generated by amplification on the genomic DNA of A. Niger using the EMBO J., Vol. 3, 1581-1585 (1984)). -1 libraries were selected. A positive EMBL4 clone that hybridized to the DIG probe was isolated from the BO-1 library, and then a 4.0 kb SpeI fragment containing the AMG gene was subcloned from the EMBL4 clone to generate plasmid pJRoy17a. Cloned into.
[0098]
3. Construction of A Niger AMG's disruption cassette
A 2.3 kb SpeI-XhoI fragment containing pyrG was isolated by gel from pJRoy10 and its restriction ends were filled with Klenow polymerase. The fragment was inserted into the BglIII site of pJRoy17 which cuts within the AMG gene to generate plasmid pSM0127 (FIG. 9). Between the two SpeI sites of pSM0127a is a 2.3 kb pyrG gene flanked by 2.2 kb 5 'AMG and 2.3 kb 3' AMG.
[0099]
4). A Niger's strain divided to obtain AMG: Construction of SM0110
A-Niger's JRoyP3 is a natural pyrG mutant of A-niger BO-1, which was selected and grown on plates containing 5'-fluoro-orotic acid (5'-FOA). It was. Since the pyrG gene encodes orotidine 5'-phosphate carboxylase, the deletion mutant can be characterized as a uridine-requiring strain. The identity of the pyrG mutant was confirmed by the fact that growth in minimal medium was complementary to the Ay Nidurans pyrG gene.
[0100]
20 μg of plasmid pSM0127 was digested with SpeI. The DNA was resolved on a 0.8% agarose gel and 6 kb consisting of a linear cleavage cassette was isolated on the gel. Strain JRoyP3 was transformed with the linear DNA. Next, genomic DNA was prepared from 200 transformants digested with SpeI. The DNA divided by the gel was transferred to a hybond nylon filter to form a hybrid with a non-radioactive DIG probe consisting of an AMG open reading frame. By inserting the split cassette into the AMG locus, the wild-type 4 kb AMG band increased to 6.3 kb. This increase is due to the 2.3 kb pyrG gene. One transformant # 110 having the above characteristics was selected and further analyzed.
[0101]
Transformant # 110 was grown in a 25 ml shake flask containing YPM medium. Strain BO-1 and parent strain JRoyP3 were grown as controls for AMG production. After 3 days, 30 μl of the clear supernatant was run on an 8-16% SDS PAGE Novex gel. Although no AMG bands were seen in transformant # 110, a number of AMG bands were produced in the positive control strain BO-1 and the parental strain JRoyP3. Transformant # 110 was named SM0110.
[0102]
Expression of Talaromyces emersonii AMG in Aspergillus oryzae and Aspergillus niger
Strains of JaL228 and SM0110 were transformed with pJaL518 as described in Christensen et al., Biotechnology, Vol. 6, pp. 1419-1422, 1988. In general, A. oryzae mycelium was grown in eutrophic broth. The mycelium was separated from the broth by filtration. The enzyme preparation Novozyme® (Novo Nordisk) was added to 1.2 M MgSO buffered to pH 5.0 with sodium phosphate._Four Was added to the mycelium in a buffer that was stabilized by infiltration, such as.
[0103]
The resulting suspension was incubated for 60 minutes at 37 ° C. with stirring. Protoplasts were filtered through Miracloth to remove mycelial debris. Harvesting the protoplasts, STC (1.2 M sorbitol, 10 mM CaCl₂, 10 mM Tris-HCl pH 7.5). Finally, protoplasts were resuspended in 200-1000 μl STC.
[0104]
For transformation, 5 μg of DNA is added to 100 μl of protoplast suspension and then 200 μl of PEG solution (60% PEG4000, 10 mM CaCl 2₂, 10 mM Tris-HCl, pH 7.5) was added and the mixture was incubated at room temperature for 20 minutes. Protoplasts were harvested and washed twice with 1.2 M sorbitol. Finally, the protoplasts were resuspended in 20 μl 1.2 M sorbitol, plated on selective plates [minimal medium + 10 g / l Bacto-Agar (Difco)] and then incubated at 37 ° C. When grown at 37 ° C. for 3-4 days, stable transformants appear as colonies that grow vigorously and form spores. Transformants were isolated twice with spores.
[0105]
Transformants were grown for 4 days at 30 ° C. in 100 ml YPM medium (2 g / l yeast extract, 2 g / l peptone and 2% maltose) in shake flasks. The supernatant was tested for AMG activity as previously described and then analyzed on an SDS PAGE gel (FIG. 10).
[0106]
Example 7 Talaromyces emersonii AMG for expression in yeast
Of four introns from the DNA sequence of Escherichia coli
For each exon, a PCR reaction was performed using a primer containing an overlapping portion relative to the adjacent exon. Tal1 and Tal4 contain an overlapping portion with the yeast vector pJSO026. Exon 1: Tal1 was used as a 5 'primer and Tal5 was used as a 3' primer and the genomic sequence encoding AMG was used as a template. Exon 2: Tal6 was used as a 5 'primer and Tal7 was used as a 3' primer and the genomic sequence encoding AMG was used as a template.
[0107]
Exon 3: Tal8 was used as a 5 'primer and Tal9 was used as a 3' primer and the genomic sequence encoding AMG was used as a template. Exon 4: Tal10 was used as a 5 'primer and Tal11 was used as a 3' primer and the genomic sequence encoding AMG was used as a template. Exon 5: Tal12 was used as the 5 'primer, Tal4 was used as the 3' primer, and the genomic sequence encoding AMG was used as the template.
[0108]
A final PCR reaction was performed to bind the five exons to a sequence containing the complete coding sequence. In this PCR reaction, the five fragments from the first PCR reaction were used as templates, and Tal1 was used as the 5 'primer and Tal4 was used as the 3' primer.
Using this last PCR fragment containing the coding region, with restriction enzymes SmaI (or BamHI) and XbaI (to remove the coding region and simultaneously create an approximately 20 bp overlap at each end to allow recombination events) In vivo recombination was performed in yeast using the cleaved pJS026.
[0109]
Tal 1: 5′-CAA TAT AAA CGA CGG TAC CCG GGA GAT CTC CAC CATG GCG TCC CTC GTT G-3 ′ (SEQ ID NO: 23);
Tal 4: 5′-CTA ATT ACA TCA TGC GGC CCT CTA GAT CAC TGC CAA CTA TCG TC-3 ′ (SEQ ID NO: 24);
Tal 5: 5'-AAT TTG GGT CGC TCC TGC TCG-3 '(SEQ ID NO: 25);
Tal 6: 5′-CGA GCA GGA GCG ACC CAA ATT ATT TCT ACT CCT GGA CAC G-3 ′ (SEQ ID NO: 26);
[0110]
Tal 7: 5'-GAT GAG ATA GTT CGC ATA CG-3 '(SEQ ID NO: 27);
Tal 8: 5′-CGT ATG CGA ACT ATC TCA TCG ACA ACG GCG AGG CTT CGA CTG C-3 ′ (SEQ ID NO: 28);
Tal 9: 5′-CGA AGG TGG ATG AGT TCC AG-3 ′ (SEQ ID NO: 29);
Tal 10: 5′-CTG GAA CTC ATC CAC CTT CGA CCT CTG GGA AGA AGT AGA AGG-3 ′ (SEQ ID NO: 30);
Tal 11: 5'-GAC AAT ACT CAG ATA TCC ATC-3 '(SEQ ID NO: 31);
Tal 12: 5'-GAT GGA TAT CTG AGT ATT GTC GAG AAA TAT ACT CCC TCA GAC G-3 '(SEQ ID NO: 32)
[0111]
Example 8 FIG. Expression of Talalomyces emersonii glucoamylase in yeast
A yeast expression vector pJS026 was constructed as described in the “Materials and Methods” section above to express A. thalomyces emersonii AMG in the yeast Saccharomyces cerevisiae YNG318.
[0112]
The yeast was transformed with pJS026 containing the DNA sequence encoding Talalomyces AMG by standard methods (see Sambrooks et al., “Molecular Cloning: A Laboratory Manual” 2nd edition, 1989, Cold Spring Harbor).
The resulting yeast cells were grown in Sc-ura medium for 3 days at 30 ° C., followed by 3 days in YPD. The culture was then centrifuged and the supernatant was then used for the thermal stability assay described in the “Materials and Methods” section.
[0113]
Thermal stability of Talaromyces AMG expressed in yeast at 68 ° C
Using the fermentation broth of Talalomyces emersonii AMG expressed in yeast (Saccharomyces cerevisiae YNG318), using the method described above in the section “Measurement of thermal stability II”, the thermal stability at 68 ° C. It was measured. The test results are shown in FIG.
[0114]
Example 9 Recombinant Talaromyces produced using A-Niger HowB112
Purification of AMG
200 ml of culture broth obtained by fermenting A. niger HowB112 carrying the Talaromyces emersonii gene was centrifuged at 9000 rpm and then dialyzed overnight against 20 mM NaOAc (pH 5). The solution was then injected onto an S Sepharose column (200 ml) that had been pre-equilibrated in 20 mM NaOAc solution (pH 5). Glucoamylase was collected in the eluate and then injected onto a Q Sepharose column (50 ml) that had been pre-equilibrated in 20 mM NaOAc (pH 4.5). Unbound material was washed away from the Q Sepharose column and then the glucoamylase was eluted using an amount exceeding 10 column volumes of a linear gradient of 0-0.3 M NaCl in 20 mM NaOAc.
[0115]
When the purity of the glucoamylase fraction was checked by SDS-PAGE, only one single band was seen. Its molecular weight was again found to be about 70 kdal, as seen for wild-type glucoamylase. When specific activity against maltose was measured, specific activities of 8.0 AGU / mg (37 ° C.) and 21.0 AGU / mg (60 ° C.) were found based on the data for the wild-type enzyme.
[0116]
Example 10 Kinetic parameters
Kinetic parameters of maltose and isomaltose hydrolysis reactions by Aspergillus niger AMG and recombinant Tallalomyces emersonii AMG expressed in Aspergillus niger.
[0117]
[Table 3]

[0118]
[Table 4]

[0119]
Example 11 Recombinant Talaromyces Emersonii A produced in A Niger
Saccharification performance of MG
The saccharification performance of Talalomyces emersonii glucoamylase was tested at various temperatures with and without the addition of acid α-amylase and pullulanase. The saccharification reaction was performed under the following conditions.
Substrate: 10DE maltodextrin, about 30% DS (w / w)
Temperature: 60, 65 or 70 ° C
Initial pH: 4.5
[0120]
Enzyme addition amount:
Recombinant Talaromyces emersonii glucoamylase produced in A. niger: 0.24 or 0.32 AGU / g DS
Acid niger-derived acid α-amylase: 0.020AFAU / g DS
Pullulanase from Bacillus bacteria: 0.03PUN / g DS
When using only Talaromyces AMG, it was added at a high addition amount (0.32 AGU / g DS), and in other cases, it was added at a low addition amount, ie 0.24 AGU / g DS.
[0121]
Saccharification
The substrate used for saccharification was prepared by dissolving maltodextrin (prepared from regular corn) in boiling Milli-Q water and adjusting the dry matter to about 30% (w / w). The pH was adjusted to 4.5 (measured at 60 ° C.). A portion of the substrate corresponding to 150 g dry solids was transferred to a 500 ml blue cap glass flask and placed in a hot water bath at the respective temperature with stirring. Enzyme was added and then the pH was readjusted as necessary (measured at the incubation temperature). Samples were taken periodically and analyzed by HPLC to determine carbohydrate composition.
[0122]
The glucose produced during saccharification is shown in the following table, the left three columns indicate saccharification by glucoamylase, acid α-amylase and pullulanase, and the right three columns indicate saccharification by glucoamylase alone. The number is% DP1 with respect to DS.
[0123]
[Table 5]

[0124]
Using an enzyme loading of 0.24 AGU / g DS corresponding to 0.03 mg / g DS, a glucose yield greater than 95% was obtained after 72 hr. A typical amount of AMG niger AMG is 0.18 AGU / g DS, which corresponds to 0.09 mg / g DS, in which case glucose is obtained in a yield of 95-96%. Therefore, the amount of enzyme added to Talalomyces AMG mg enzyme protein required in the saccharification method is significantly lower than that of A. niger AMG because of the high specific activity of tea-emersonii AMG.
[0125]
Example 12 FIG. Recombinant Talalomyces emersonii AMG expressed in yeast
Temperature stability -T ¹ / ₂ (Half life)
The thermostability of recombinant Tallalomyces emersonii glucoamylase expressed in yeast (purified using the method described in Example 9) is described in the section “Materials and Methods”. T¹/₂ (Measurement of (half-life)] "was measured at 70 ° C, pH 4.5, 0.2 AGU / ml using the method described above.
FIG. 13 shows the test results. Recombinant Tallalomyces emersonii glucoamylase T expressed in yeast¹/₂ Was about 110 minutes at 70 ° C. as a result of measurement.
[Brief description of the drawings]
FIG. 1 shows an SDS-PAGE gel (stained with Coomassie blue) used to determine the molecular weight (Mw) of the purified Talaromyces emersonii CBS 793.97 glucoamylase of the present invention.
1: Standard marker
2: Pool of Q Sepharose (1. Run)
3: S Sepharose pool
FIG. 2 shows a graph of the pH-activity of Talaromyces emersonii and Aspergillus niger glucoamylase (AMG) in 0.5% maltose at 60 ° C.
FIG. 3 shows a graph of temperature-activity of Talaromyces emersonii CBS 793.97 glucoamylase versus Aspergillus niger glucoamylase (AMG).
FIG. 4 shows Talomyces emersonii CBS 793.97 glucoamylase versus Aspergillus niger glucoamylase (AMG) 50 mM NaOAc, 0.2 AGU / ml, pH 4.5 at 70 ° C.¹/₂ The curve for measuring (half-life) is shown.
FIG. 5 shows the sequence of the AMG locus of Talaromyces emersonii. The predicted amino acid sequence is shown below the nucleotide sequence. Four introns are shown in lower case. The consensus intron sequence is underlined. The putative signal and propeptide are two underlined and dotted underlined respectively.
FIG. 6 shows ANIGER's AMG (An amg-1. pro), A oryzae AMG (Ao AMG. Pro), and the amino acid sequence of Talaromyces emersonii AMG (Tal-AMG. Pro). The same amino acid is indicated by *. The signal peptide and propeptide are each underlined by one and two underlines.
FIG. 7 shows the Aspergillus fungus expression cassette pCaHj483 used in Example 5.
FIG. 8 shows the Aspergillus fungus expression plasmid pJa1518 used for the gene for AMG of Talalomyces emersonii.
FIG. 9 shows the construction of A. Niger's split plasmid.
FIG. 10 shows two transformants JaL228 # 5.77 and HowB112 # 8.10 SDS Page gels that express the Tallomyces emersonii glucoamylase of the present invention. JaL228 and HowB112 are untransformed parental strains. Mw is the molecular weight of Progma protein.
FIG. 11 shows strain A. The heat stability of AMG of tea emersonii produced by Niger HowB112 (measured at 50 mM NaOAc (pH 4.5), 70 ° C., 0.2 AGU / ml) is shown. As a result of measurement, T¹/₂ Was 20 minutes.
FIG. 12 is a diagram comparing the heat stability at 68 ° C. of the fermentation broth of tea Emersony AMG and A. niger AMG produced in yeast and expressed in yeast.
FIG. 13 shows the measurement test results of the thermal stability of recombinant Tallalomyces emersonii AMG produced in yeast at 70 ° C., pH 4.5, 0.2 AGU / ml.¹/₂ Was about 110 ° C.
[Sequence Listing]

Claims (30)

An enzyme having the following properties and having glucoamylase activity:
(1) The molecular weight measured by SDS-PAGE is about 70 kDa;
(2) when measured at a temperature of 60 ° C., the optimum pH is 4.5 ;
(3) When measured at pH 4.3, the optimum temperature is 70 ° C;
(4) The half-life (T1 / 2) is at least 100 minutes when measured at pH 4.5 and a temperature of 70 ° C .;
(5) the isoelectric point is less than 3.5; and (6) derived from the species Talaromyces emersonii.   The enzyme having glucoamylase activity according to claim 1, which is derived from Talaromyces emersonii CBS 793.97.   An enzyme having glucoamylase activity, comprising the amino acid sequence set forth in SEQ ID NO: 7.   Comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7 and having a half-life of at least 100 minutes (T1 / 2) An enzyme having glucoamylase activity. SEQ ID NO: 33 is encoded by hybridizing nucleic acid under high stringency conditions to a formed Ru nucleic acids from a nucleotide sequence complementary to the nucleotide sequence set forth in, and measured at a temperature of pH 4.5 and 70 ° C. An enzyme with glucoamylase activity having a half-life (T1 / 2) of at least 100 minutes.   A nucleic acid encoding the enzyme according to any one of claims 3 to 5. SEQ ID NO: 33 formed Ru the nucleotide sequence set forth in, the nucleic acid of claim 6.   A vector comprising the nucleic acid according to claim 6 or 7.   The vector according to claim 8, which is an expression vector.   A host cell transformed with the vector according to claim 8 or 9. A method for producing the enzyme according to claim 1 or 2,
(1) culturing a microorganism having the ability to produce the enzyme and belonging to the species Talaromyces emersonii; and (2) collecting the enzyme from the culture;
A method comprising that. The Talaromyces & Emerusonii (Talaromyces emersonii) species, Ru Oh in Talaromyces-Emerusonii (Talaromyces emersonii) CBS 793.97 strain, The method of claim 11. A method for producing the enzyme according to any one of claims 3 to 5,
(1) culturing the host cell according to claim 10; and (2) collecting the enzyme from the culture;
A method comprising that.   A method for converting starch or partially hydrolyzed starch into a syrup containing dextrose; in the presence of glucoamylase according to any one of claims 1-5, A method comprising the step of saccharification.   15. The method according to claim 14, wherein the amount of glucoamylase added is in the range of 0.05 to 0.5 AGU per gram of dry solid.   16. The method according to claim 14 or 15, comprising saccharifying starch hydrolyzate of at least 30% by weight of the dry solid.   The method according to any one of claims 14 to 16, wherein the saccharification step is performed in the presence of a debranching enzyme selected from the group of pullulanase and isoamylase.   18. The method according to claim 17, wherein the pullulanase is a pullulanase derived from Bacillus acidopullulyticus, Bacillus deramificans or Pseudomonas amyloderamosa.   The method according to any one of claims 14 to 18, wherein the saccharification step is performed at a pH of 3 to 5.5 and a temperature of 60 to 80 ° C.   The method according to any one of claims 14 to 18, wherein the saccharification step is performed at 63 to 75 ° C for 24 to 72 hours.   The method according to any one of claims 14 to 18, wherein the saccharification step is performed for 36 to 38 hours at a pH of 4 to 4.5.   A method for saccharifying a liquefied starch solution, comprising a saccharification step using the glucoamylase according to any one of claims 1 to 5.   Use of the glucoamylase according to any one of claims 1 to 5 in a starch conversion method.   Use of the glucoamylase according to any one of claims 1 to 5 in a continuous starch conversion method.   Use of the glucoamylase according to any one of claims 1 to 5 for a method for producing an oligosaccharide.   Use of the glucoamylase of any one of Claims 1-5 for the manufacturing method of special syrups.   Use of the glucoamylase according to any one of claims 1 to 5 in a method for producing ethanol for fuel.   Use of the glucoamylase of any one of Claims 1-5 for the manufacturing method of a favorite drink.   Use of the glucoamylase according to any one of claims 1 to 5 in a fermentation method for producing an organic compound.   30. Use according to claim 29, wherein the organic compound is citric acid, ascorbic acid, lysine or glutamic acid.

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